Androgens are required for maintenance of spermatogenesis and for stimulation of the accessory glands. The peripheral androgen concentration is determined mainly by the steroidogenic activities of Leydig cells which are regulated by luteinizing hormone (LH), other hormones and also by locally produced factors. The androgen level in the testis is also, therefore, under the influence of LH. Since Leydig cells are the source of androgens, much higher levels are present in the testis than in peripheral androgen-dependent organs. There has been a long-running debate as to whether this high intratesticular concentration of androgen is essential for maintenance of spermatogenesis, or whether it is a prerequisite for the secretion of androgens. It has been suggested that locally produced factors operate to keep intratesticular testosterone levels high, especially in between LH pulses or at those specific stages of spermatogenesis which require high testosterone levels (Sharpe, 1984, 1986). Support for this hypothesis was

ABSTRACT

The presence of non-specific esterase activity is correlated with different Leydig cell characteristics: 3β-hydroxysteroid dehydrogenase (3β-HSD), human chorionic gonadotrophin binding and LH-stimulated steroid production. This indicates that esterase can be used as a marker enzyme for Leydig cells. Esterase, however, has also been used as a marker enzyme for macrophages. We have compared, using biochemical and histochemical techniques, the esterase activity of Leydig cell preparations from mature and immature rats and of preparations enriched in testicular or peritoneal macrophages. Leydig cells were identified by staining for 3β-HSD, and macrophages by phagocytosis of fluorescent beads. Leydig cell preparations from mature rats showed an approximately 400-fold higher esterase activity than peritoneal macrophage preparations and an approximately 50-fold higher activity than testicular macrophage preparations. Leydig cell preparations from mature rats showed a 60-fold higher esterase activity than Leydig cell preparations from immature rats.

Differences in esterase activity were also demonstrated histochemically. Leydig cells from mature rats showed positive esterase staining after 30 s at room temperature. Testicular macrophages showed esterase activity after staining for 3 min. Only approximately 25% of the 3β-HSD-positive cells from immature rats showed esterase activity after staining for 6 min. Esterase is therefore a useful marker enzyme for Leydig cells from mature rats and can be of help in studies concerning the development of these cells.

J. Endocr. (1986) 108, 329–334

ABSTRACT

The formation of new Leydig cells in adult male rats was studied after the complete destruction of the original population by ethane dimethane sulphonate (EDS). Following administration of EDS, proliferating interstitial cells were labelled in a pulse-chase experiment by way of three [³H]thymidine injections on days 2, 3 and 4 after EDS administration. Some of the newly formed Leydig cells found 14 days after EDS administration were labelled with [³H]thymidine, indicating that these Leydig cells were derived from precursor cells, most likely mesenchymal cells, that had incorporated [³H]thymidine at days 2, 3, or 4 after EDS administration. At 21 days after EDS administration, the total number of Leydig cells (labelled plus unlabelled) had increased 7- to 16-fold compared with the number of cells that were present 14 days after EDS had been administered.

In a second series of experiments, [³H]thymidine was given 2 h before the rats were killed (short-term labelling experiment). In this experiment it was shown that the proliferative activity of the mesenchymal cells, which are presumed to be the precursors of the Leydig cells, after a considerable increase at day 2 after EDS administration, had returned to the control level at day 7. However, the total number of mesenchymal cells (labelled plus unlabelled) remained increased from 2 to 49 days after EDS administration. This indicated that the majority of the new Leydig cells which were formed from day 14 onwards probably did not derive from differentiating mesenchymal cells. The labelling index of the Leydig cells was approximately 100 times higher 21 days after EDS administration than that of the untreated controls, showing that many Leydig cells were formed by proliferation of the newly formed Leydig cells. Thereafter, the labelling index of the Leydig cells gradually decreased, whereas the total number of Leydig cells still increased threefold. At 49 days after EDS administration, the number of Leydig cells was approximately 80% of that in normal adult rats.

It is concluded that the regeneration of Leydig cells after EDS administration is the result of two successive waves of proliferation, namely of the precursor cells (mesenchymal cells) and of the newly formed Leydig cells.

Journal of Endocrinology (1990) 126, 229–236

ABSTRACT

Several studies have shown that the cytotoxic agent ethane-1,2-dimethane sulphonate (EDS) specifically destroys Leydig cells in the adult rat testis. It has also been reported that when rats are pretreated with human chorionic gonadotrophin (hCG), administration of EDS does not result in the complete destruction of the Leydig cell population. It has been suggested that hCG pretreatment 'protects' Leydig cells against the cytotoxic action of EDS. In the present study the underlying principles for this resistance to the cytotoxic effects of EDS have been investigated. Within 48 h of the start of daily hCG treatment the number of nuclear profiles of Leydig cells (henceforth called relative number of Leydig cells) had increased from 1014 ± 40 to 1368 ± 30 cells per 1000 Sertoli cell nuclei. Previous experiments have indicated that these newly formed Leydig cells probably develop from differentiating Leydig cell precursors. When EDS is administered concomitantly with the third injection of hCG (2 days after the start of hCG treatment), the relative number of Leydig cells surviving EDS treatment was 388 ± 52 per 1000 Sertoli cells. Hence, there is a similarity between the increase in the relative number of Leydig cells after 2 days of hCG treatment and the relative number of EDS-resistant Leydig cells. The Leydig cells that survived EDS administration showed characteristics which also occur in developing Leydig cells in the immature testis. It is concluded that, in rats pretreated with hCG for 2 days before EDS administration, new Leydig cells with some immature characteristics are formed. One of these characteristics is that these cells are insensitive to EDS.

Journal of Endocrinology (1992) 134, 85–90

ABSTRACT

The biochemical activities involved in the maintenance of Leydig cell functions, and the effects of hypophysectomy and human chorionic gonadotrophin (hCG) on these functions are largely unknown. In the present study, adult hypophysectomized rats were used as a model to determine the effects of these treatments on a number of biochemical and morphological parameters. After 33 days of hypophysectomy, the morphology of the Leydig cells had been drastically altered. In addition, α-naphthol and β-naphthol esterase activity as well as the steroidogenic capacity of the Leydig cells were greatly reduced at this time. In contrast, the level of sterol carrier protein 2 (SCP₂), a Leydig cell-specific protein, was affected by hypophysectomy much less than the other parameters measured. Two daily injections of hCG to rats hypophysectomized for 31 days resulted in no change in the morphology of the Leydig cells, or in their proliferative activity. Non-specific esterase activities were also unaffected by 2 days of treatment with hCG. However, two injections of hCG to rats hypophysectomized for 31 days resulted in nearly complete restoration of steroidogenic capacity, and a 3·5-fold increase in the level of SCP₂. These findings indicate that hypophysectomy results in significant morphological and biochemical changes in Leydig cells, and that hCG is capable of restoring some of these capacities within a short time.

Journal of Endocrinology (1990) 126, 367–375

ABSTRACT

In testis tissue from mature rats the non-specific lipid transfer protein (nsLTP), also called sterol carrier protein₂ (SCP₂), is concentrated in the Leydig cells and cannot be detected in Sertoli cells or germinal cells. Conclusions were reached after cell fractionation studies with normal testis tissue and after selective destruction of Leydig cells or germinal cells in vivo.

The amount of nsLTP (SCP₂) in testis tissue increased twofold 48 h after two daily injections of human chorionic gonadotrophin (100 i.u., s.c.) and decreased twofold after plasma luteinizing hormone levels were suppressed to almost undetectable levels with silicone elastomer implants containing testosterone.

The specific localization in the Leydig cells and the luteinizing hormone-dependent cellular concentration of nsLTP/SCP₂ support the possibility that this protein could play a role in the regulation of steroidogenesis by regulating the availability of cholesterol for the P₄₅₀ side-chain cleavage enzyme in the mitochondria of Leydig cells.

ABSTRACT

After the addition of charcoal-treated testicular fluid to Leydig cells isolated from 22-day-old rats, pregnenolone production could be increased to a maximum of tenfold within 30 min in a dose-dependent manner. Testicular fluid, but not serum, further increased pregnenolone formation threefold when pregnenolone production by Leydig cells was stimulated by the addition of LH-releasing hormone (fourfold), LH (25-fold) and 22R-hydroxycholesterol (300-fold). The effect of testicular fluid on steroid production in the presence of 22R-hydroxycholesterol was not inhibited by cycloheximide whereas cycloheximide completely inhibited the effect of LH. It appears unlikely that steroids, lipoproteins or other plasma components constitute the stimulatory agents in testicular fluid. The biologically active principles may be locally produced factors with a molecular weight > 25 000. Similar biological activities could be shown in testicular lymph from boars but not in systemic lymph from boars nor in charcoal-treated bovine follicular fluid. The presumably locally produced factor(s) may amplify the effect of LH and can thus act as a local modulator(s).

J. Endocr. (1986) 109, 111–117

Abstract

Effects of FSH on ovarian follicular development can be modulated by factors present in serum or by locally produced factors in follicular fluid. Some of these factors may act directly on the FSH receptor. A Chinese hamster ovary cell line (CHO-F3B4) stably transfected with the human FSH receptor has been used to measure the effects of these modulators on FSH-stimulated adenylate cyclase activity. After incubation of CHO-F3B4 cells with human recombinant FSH (recFSH) for 4 h, cAMP levels were elevated 100–230 times above basal levels (ED₅₀ 24·9 mU/ml recFSH). cAMP production was inhibited after the addition of increasing amounts (up to 90% of the incubation volume) of hypogonadotrophic human serum (HS) at a fixed stimulatory dose of 30 mU/ml recFSH. At 10% HS the cAMP response was diminished to approximately 40–60% of the original value, whereas at a concentration of 90% HS the cAMP values were diminished to 30%. Effects of serum components on cell viability could be excluded, since forskolin- and cholera-toxin-stimulated cAMP production were not affected by pre-incubation of the cells in the presence of HS. The FSH-stimulated oestradiol production in rat Sertoli cells, which has been used frequently for in vitro bioassays of FSH, was almost completely inhibited by the addition of human serum, suggesting that serum has more pronounced effects on events downstream of receptor activation. Various specific FSH binding inhibitors have been demonstrated by radioreceptor assays to be present in serum. In order to assess whether such FSH receptor binding inhibitors would also inhibit receptor activation, the specific conditions used in the radioreceptor assays (buffers of low ionic strength) were also used to measure the effects of serum on FSH receptor activation. Under these conditions (a low-salt buffer, corrected for low osmolarity with 200 mm sucrose), CHO-F3B4 cells responded to FSH stimulation in a similar way to that observed in normal buffers. When CHO-F3B4 cells were incubated in this low-salt buffer with a fixed low dose of FSH (3 mU/ml), the addition of 3–90% (v/v) dialysed HS inhibited the FSH-stimulated cAMP accumulation to a similar extent to that in standard conditions. The observed inhibition of adenylate cyclase activation by the low-molecular-mass fraction (<10 kDa) of HS could be attributed to the presence of salts in this fraction, since the addition of PBS in similar concentrations displayed an equal degree of inhibition.

It is concluded that the inhibitory effects of serum on FSH-stimulated cAMP production in CHO-F3B4 cells are small, compared with the inhibition of aromatase induction in rat Sertoli cells. The strong inhibition of aromatase in rat Sertoli cells may result from the effects of serum acting on the FSH receptors as well as on other pathways not related to the FSH receptor. Therefore, measurement of aromatase in Sertoli cells is not suitable for the detection of inhibitors of FSH receptor activation. The CHO-F3B4 cells are useful for the measurement of whether inhibition of FSH receptor activation occurs in serum or follicular fluid from patients with disturbed follicle development.

Journal of Endocrinology (1996) 150, 505–514

ABSTRACT

The effect of 100IU human chorionic gonadotrophin (hCG) on testicular capillary blood flow was studied in adult male rats using a ¹³³Xe clearance method and a radioactive microsphere technique. To investigate the role of Leydig cells in regulation of testicular blood flow after treatment with hCG, rats were pre-treated with ethane dimethylsulphonate (EDS) which selectively destroys mature Leydig cells. Six hours after treatment with hCG, testicular blood flow decreased in control and hypophysectomized rats to 25–50% of normal values, but not in EDS-pretreated animals. Prostaglandin E₂ levels were also determined 6 h after an injection of hCG. A 300-fold increase in the concentration of prostaglandin E₂ occurred in normal testis tissue. This rise was markedly inhibited if EDS was given 3 days before administration of hCG. Furthermore, 6 h after administration of hCG, the filling of the testicular capillary bed with methylacrylate was decreased, while in control rats and rats treated with EDS and hCG, complete filling of the capillaries was seen. Cell degeneration in some subcapsular seminiferous tubules was observed 6–10 days after treatment with hCG.

The results suggest that the hCG-induced precapillary vasoconstriction, probably mediated (in part) by prostaglandins, causes reduction in testicular blood flow 6 h after administration of hCG, and may result in cell damage.

J. Endocr. (1988) 117, 51–57

ABSTRACT

The effects of pure FSH and/or LH preparations on the number of Leydig cells and their function in immature hypophysectomized rats have been investigated. As a result of hypophysectomy at the age of 17–18 days, the number of recognizable Leydig cells per testis decreased, as did the steroidogenic capacity in vivo and in vitro. Treatment with 64 μg FSH on both 22 and 23 days of age, did not affect the number of recognizable Leydig cells. In contrast, two injections of LH (10 μg) caused a sixfold increase in the number of Leydig cells, but had a negative effect on spermatogenesis. These stimulatory and inhibitory effects of LH diminished when FSH was added. Treatment with FSH for 7 days caused a twofold increase in the number of Leydig cells when compared with hypophysectomized controls. 3β-Hydroxysteroid dehydrogenase (3β-HSD) and esterase activity in Leydig cells also increased under the influence of FSH. The pregnenolone production per Leydig cell in the presence of 5-cholesten-3β,22(R)-diol (22R-hydroxycholesterol) as substrate showed a sevenfold increase. Plasma testosterone levels 2 h after injection of human chorionic gonadotrophin in intact rats and hypophysectomized FSH-treated rats were the same. Following LH treatment for 7 days, the number of Leydig cells proved to be 11 times higher, and 3β-HSD and esterase activity were not different from intact controls. The testicular pregnenolone production was four- to fivefold higher when compared with untreated hypophysectomized rats. However, pregnenolone production per Leydig cell in LH-treated rats was only slightly different from the hypophysectomized controls.

In conclusion, FSH treatment caused an increase in the number and steroidogenic activity of Leydig cells, and LH had a major effect on the number of Leydig cells, but did not stimulate the steroidogenic capacity.

Journal of Endocrinology (1989) 120, 97–106